Methods and materials for the reproducible generation of high producer cell lines for recombinant proteins

Abstract

The invention lies in the field of production of recombinant gene products in eukaryotic cells. The invention refers to methods and materials for the fast and reproducible generation of production cells lines suitable for large scale production of recombinant gene products. The invention encompasses specific vector systems, genetic engineered host-cells and methods of use.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is the U.S. national stage of International Patent Application No. PCT / EP2009 / 002266, filed on Mar. 27, 2009 and entitled “METHODS AND MATERIALS FOR THE REPRODUCIBLE GENERATION OF HIGH PRODUCER CELL LINES FOR RECOMBINANT PROTEINS,” which claims the benefit of priority from US Provisional Patent Application No. 61 / 040,315, filed on Mar. 28, 2008, and from European Patent Application No. 08006023.9, also filed on Mar. 28, 2008. The disclosures of the foregoing applications are incorporated herein by reference in their entirety.SEQUENCE LISTING[0002]The entire content of a Sequence Listing titled “Sequence_Listing.txt,” created on Aug. 16, 2010 and having a size of 0.5 kilobytes, which is being submitted herewith in electronic form in connection with the present application, is hereby incorporated by reference herein in its entirety.[0003]1 . Field of the Invention[0004]The invention lies in the field of production of recomb...

AI Technical Summary

Benefits of technology

The invention provides a new way to create cells that can produce large amounts of recombinant proteins or nucleic acids. This method allows for the rapid and consistent generation of these cells, which can be useful for research and development. Overall, the invention provides a faster and more reliable way to create reliable host cells for producing desired gene products.

Problems solved by technology

Although several hundreds of single cell derived clones are analyzed, the expression level was often still weak, which requires the optimization of transfection procedures or the amplification of initially integrated copies of the expression construct, e.g. by increasing concentration of methotrexate or another amplifying system.

The major problem with this approach is the random nature of the integration event.

Due to the stochastic nature of this non-homologous integration process, the integration cannot be directed to loci with a high transcriptional activity (transcriptional hot spots).

In order to use these specific recombinases for the generation of a production cell line, the major problem is to identify a suitable gene for insertion of recognition sites by homologous recombination.

Although applicable, these approaches are restricted to known, non-essential, highly expressed cellular genes in the respective host-cell.

Therefore the use of this approach in host from which limited sequence information (like hamster) is available, will limited its applicability.

Thus, this document does not provide a reliable method for obtaining high-producing host cells.

This results in a decreased stability of the producer cell line.

In addition the random selected locus might have a good transcriptional activity, but it might not support efficient recombination by the used recombinase.

A further potential obstacle in the use of such recombinases is the presence of pseudo-recognition sites within the genome of the desired host-cell.

These pseudo-recognition sites could lead to unwanted recombination events.

This will lead to a reduce efficiency of the system and to potentially genetic modified cells with undesirable phenotype.

Although this approach deems functional, its efficiency and universal applicability in the generation of a high-yield production cell will be low.

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